Urogenital carcinoma tlp complex peptides and antibodies thereof

ABSTRACT

It is described a peptide comprising a specific urogenital carcinoma TLP epitope, wherein the TLP comprises the aminoacid sequence: GlyProProGluValGlnAsnAlaAsn. Also described are reagents to detect said urogenital TLPs and pharmaceutical compositions.

[0001] The present invention concerns peptides of the TLP complex(tumour released proteins) isolated from urogenital apparatus carcinoma.

[0002] In particular the invention refers to peptides of TLP proteincomplexes from human urogenital apparatus carcinoma having antigenicactivity and to antibodies able to react with them, to be used indiagnostics and clinics.

[0003] TLP complexes are protein complexes which are present in humantumoral cells. Among TLP proteins a 214 KDa protein is described (TarroG., Oncology 40, 248-253, 1983). TLP are isolated from tumoral tissuesas described in the European Patent EP 283443. Italian PatentApplication No. RM92A000506 identifies a TLP protein from lungcarcinoma. The author of the instant invention has surprisingly foundthat TLP from urogenital apparatus carcinoma comprise new peptideshaving sequences which differ from known TLP peptides.

[0004] Therefore there is the need to identify TLP peptides, acting asepitopes, from urogenital carcinoma, to produce specific reagents, asantibodies.

[0005] The author of the instant invention has identified a peptidehaving a sequence comprised in the sequence of the 214 kDa TLP proteinfrom either cervix uterus or testis adenocarcinoma, and from kidneyneoplasia.

[0006] Therefore it is an object of the instant invention a peptidecomprising a specific urogenital carcinoma TLP epitope, wherein said TLPis characterised by comprising the aminoacid sequence of SEQ ID No. 1:

[0007] GlyProProGluValGlnAsnAlaAsn.

[0008] According to a preferred embodiment the peptide comprises theaminoacid sequence of SEQ ID No. 1.

[0009] Further object of the invention are specific reagents able torecognise the TLP from urogenital carcinoma, preferably said reagentscomprise TLP antibodies. More preferably said antibodies recognise thepeptide fragment having the sequence of SEQ ID No. 1.

[0010] Further object of the invention are diagnostic kits to identifyTLP from a sample comprising as specific reagents the antibodies of theinvention.

[0011] Another object of the invention is a pharmaceutical compositioncomprising as active agent the peptide of the invention.

[0012] The invention will now be described according to exemplificatingbut not limiting examples.

EXAMPLE 1

[0013] Preparation of Tumoral Extract

[0014] A tumoral biopsy of 12.55 g, from a 45 old woman having a cervixcarcinoma, was thawed at room temperature and necrotic tissues weresurgically removed. When the material resulted to be homogeneous, manywashings were performed with Tris 1× (10 mM Tris-HCl pH 7.2) and thetissue subjected to three freeze-thawing cycles.

[0015] Used Tris washing solution was collected and centrifuged at33,000 rpm for 1 hr. The supernatant was collected and frozen.

[0016] The tissue was sonicated three times for three min. andsubsequently ultracentrifuged at 33,000 rpm for 120 min. The supernatant(5, 7 ml) was collected, filtered on Agrodisc filters (0.45 μm), and thetissue was suspended to a ratio of 1 g/ml Tris, and ultracentrifuged at33,000 rpm for 60 min. The supernatant (0.5 ml) was filtered and addedto the previous one.

[0017] The same process was performed on either testicular carcinoma(total yield 1.9 ml) or kidney neoplasia samples.

EXAMPLE 2

[0018] Identification of Peptides Comprised in the TLP Protein fromUrogenital Apparatus Carcinoma

[0019] The TLP complex is isolated from urogenital apparatus carcinomaextracts as described in the EP 283443 patent. Samples utilised are:

[0020] 1) cervix adenocarcinoma;

[0021] 2) testicular carcinoma;

[0022] 3) kidney neoplasia.

[0023] The protein content resulted to be as follows: TABLE 1 SampleTotal mg mg/ml TLP cervix 59,5 9,6 adenocarcinoma testicular  7,4 3,9carcinoma kidney neoplasia  7,4* 1,5

[0024] the TLP identification was performed through molecular weightdetermination (214 kDa) by means of denaturing electrophoretic analysis(SDS).

[0025] The removal of the main part of contaminants was performed bytaking off gel sections corresponding to an apparent molecular weight of214 kDa and by further electroeluting with a microelectroelutionapparatus (AMICON).

[0026] Subsequently the TLP purity was confirmed by denaturingelectrophoresis analysis. TLP was then transferred onto a PVDF(polyvinylidifluoride) membrane with an high protein capturing activity,and a sequential aminoacid analysis according to the Edman method wasperformed, with a protein automated sequencing apparatus by AppliedBiosystems.

[0027] The aminoacid sequence whiz was found in all of samples was thefollowing: GlyProProGluValGlnAsnAlaAsn (SEQ ID No.1).

1 1 1 9 PRT Homo Sapiens 1 Gly Pro Pro Glu Val Gln Asn Ala Asn 1 5

1. Peptide comprising a TLP urogenital specific epitope, wherein saidTLP comprises the amino acid sequence of SEQ ID No.
 1. 2. Peptideaccording to claim 1 comprising the amino acid sequence of SEQ ID NO. 1.3. Antibody able to recognise at least one peptide according to claim 1or
 2. 4. Diagnostic kit to identify TLP from urogenital tumoral samplescomprising as specific reagent the antibody according to claim
 3. 5.Process to identify TLP complexes from urogenital tumoral samplescharacterised by the fact of comprising the following steps: toimmunoprecipitate said sample with a first aliquot of immune serumagainst the peptide of claim 1 or 2; to detect said TLP complexes fromthe first immunoprecipitate by means of a reaction with a second aliquotof said immune serum and with a detecting mean of said reaction. 6.Pharmaceutical composition comprising as active agent the peptide ofclaim 1.